This invention relates to the use of the coenzyme nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), in a lactate dehydrogenase-lactate assay analytical procedure.
In the determination of enzymes and other biological constitutents, the reaction generally involve enzymes, coenzymes and substrates.
Enzymes are complex proteins with large molecular weights, and are usually of unknown chemical structure. They are classified by their substrate specificity, and catalytic activity. Enzymes are biological catalysts, which can catalyze the reaction of a single substrate, or the reaction of a group of similar substrates.
Coenzymes are organic chemicals with well-defined chemical structures. They usually have lower molecular weights then enzymes. They are required for specific enzyme assay or reaction. Coenzymes are irreversibly changed in their structure and/or atomic composition in the assay. Their reactions are stoichiometric with the substrate. With certain coenzymes having strong absorbance, the creation or disappearance of the absorbing form can be followed photometrically. For example, nicotinamide adenine dinucleotide (NAD) and reduced nicotinamide adenine dinucleotide (NADH) are used in many important clinical assays. Both species have a molecular weight of about 700. NADH absorbs strongly at 340 nm, while NAD does not.
Substrates are organic chemicals of known structure, whose reactions or interactions are catalyzed by enzymes resulting in a change in the substrate's chemical structure, atomic composition, or stereochemistry. In general, substrates are prone to degradation, both chemically and microbiologically. Substrates chemically degrade or hydrolyze in aqueous media, and serve for food for bacteria, fungi and other microorganisms. Typical substrates are glucose, lactate or lactic acid, gluconate and the like.
Because of their high specificity, the use of enzyme determinations has significantly increased during the recent years. At present, the greatest limitation on the use of enzyme reagents lies in the unstable nature of the species therein. Numerous labile components are usually involved. To complicate matters, the exact nature of enzymes, as well as the mechanisms of their action, remains unknown for the most part. Therefore, rigorous quality control measures are required to assure accurate and consistent results. Such measures can be costly.
In the prior art, to ensure strict quality control, emphasis was placed on attempts in stabilizing the labile ingredients in the reagents, i.e., to prevent them for degrading. For example, the enzyme or coenzyme is locked into a solid matrix, either by dry blending, freeze drying, or by locking the chemical structure of the enzyme on to a solid matrix. These methods are expensive, require complicated manufacturing processes, and are less convenient for the user. Product uniformity is difficult to maintain with solid reagents. It was reported that most commercial freeze dried reference serums list an acceptable bottle-to-bottle variation of enzyme constituents at .+-.10% of the mean. More importantly, the user has to bear the burden of assuring the quality control in the dilution and use of the reagent.